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Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2011 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 9, 1083-1094 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa(3) CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O(2) reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a pump site and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the HID kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from 02 reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F -> O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P -> F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F -> O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.

Place, publisher, year, edition, pages
2011. Vol. 1807, no 9, 1083-1094 p.
Keyword [en]
Respiration, Electron transfer, Cytochrome aa(3), Membrane protein, Electrostatics, Energy transduction
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-68035DOI: 10.1016/j.bbabio.2011.03.012ISI: 000293550300008OAI: oai:DiVA.org:su-68035DiVA: diva2:471828
Note

authorCount :6

Available from: 2012-01-03 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Structural elements involved in protein-mediated proton transfer: Implications from studies of cytochrome c oxidase
Open this publication in new window or tab >>Structural elements involved in protein-mediated proton transfer: Implications from studies of cytochrome c oxidase
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proton transfer is one of the most common reactions in biological systems. During energy conversion inside a cell, proton transfer is crucial to maintain an electrochemical proton gradient across the cell membrane. This gradient is in turn used to e.g. produce ATP, the energy currency of the cell. One of the key components of the build-up of this gradient is cytochrome c oxidase. This membrane-bound enzyme catalyzes the reduction of molecular oxygen to water, using protons and electrons, and in the process protons are pumped across the membrane. All protons used during oxygen reduction and those that are pumped, are transferred via hydrophilic pathways inside the hydrophobic interior of the enzyme. One of these pathways, called the D pathway, is used to transfer protons both to the catalytic site and towards a pump site. It is yet not fully understood how these proton-transfer reactions are timed, coupled and controlled.

 

This thesis is focused on studies of proton-transfer reactions through the D pathway in variants of cytochrome c oxidase that lack the ability to pump protons. The results suggest that changes in pKa values of key residues, as well as structural changes inside the pathway, can explain the non-pumping phenotypes. The results have led us to propose that an internal proton shuttle (Glu286I) can adopt two different conformations that are in equilibrium with each other, and that this equilibrium is altered in non-pumping variants of cytochrome c oxidase. We also observed that proton transfer through the D pathway could occur with the same rate as in the wild-type enzyme even when one of the key residues (Asp132I) is absent. This result contradicts previous assumptions that acidic residues must be present at an orifice of proton pathways. We therefore suggest that this specific residue could have an additional role, e.g. as a selectivity filter that excludes all ions except protons from entering the pathway.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 62 p.
Keyword
cytochrome c oxidase, proton transfer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-85934 (URN)978-91-7447-565-4 (ISBN)
Public defence
2013-02-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Accepted; Paper 3: Manuscript

Available from: 2013-01-24 Created: 2013-01-10 Last updated: 2013-03-15Bibliographically approved

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